This invention relates to methods of designing and isolating of nucleic acid molecules with desired catalytic activity, the molecules themselves and derivatives thereof
The following is a brief description of catalytic nucleic acid molecules. This summary is not meant to be complete but is provided only for understanding of the invention that follows. This summary is not an admission that all of the work described below is prior art to the claimed invention.
Catalytic nucleic acid molecules (ribozymes) are nucleic acid molecules capable of catalyzing one or more of a variety of reactions, including the ability to repeatedly cleave other separate nucleic acid molecules in a nucleotide base sequence-specific manner. Such enzymatic nucleic acid molecules can be used, for example, to target cleavage of virtually any RNA transcript (Zaug et al., 324, Nature 429 1986 ; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Research 1371, 1989). Catalytic nucleic acid molecules mean any nucleotide base-comprising molecule having the ability to repeatedly act on one or more types of molecules, including but not limited to enzymatic nucleic acid molecules. By way of example but not limitation, such molecules include those that are able to repeatedly cleave nucleic acid molecules, peptides, or other polymers, and those that are able to cause the polymerization of such nucleic acids and other polymers. Specifically, such molecules include ribozymes, DNAzymes, external guide sequences and the like. It is expected that such molecules will also include modified nucleotides compared to standard nucleotides found in DNA and RNA.
Because of their sequence-specificity, trans-cleaving enzymatic nucleic acid molecules show promise as therapeutic agents for human disease (Usman & McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037). Enzymatic nucleic acid molecules can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the RNA non-functional and abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited. In addition, enzymatic nucleic acid molecules can be used to validate a therapeutic gene target and/or to determine the function of a gene in a biological system (Christoffersen, 1997, Nature Biotech. 15,483).
There are at least seven basic varieties of enzymatic RNA molecules derived from naturally occurring self-cleaving RNAs (see Table I). Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. In general, enzymatic nucleic acids act by first binding to a substrate/target RNA. Such binding occurs through the substrate/target binding portion of an enzymatic nucleic acid molecule which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic and selective cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and thus can repeatedly bind and cleave new targets.
In addition, several in vitro selection (evolution) strategies (Orgel, 1979, Proc. R. Soc. London, B 205, 435) have been used to evolve new nucleic acid catalysts capable of catalyzing a variety of reactions, such as cleavage and ligation of phosphodiester linkages and amide linkages, (Joyce, 1989, Gene, 82, 83-87; Beaudry et al., 1992, Science 257, 635-641; Joyce, 1992, Scientific American 267, 90-97; Breaker et al., 1994, TIBTECH 12, 268; Bartel et al., 1993, Science 261:1411-1418; Szostak, 1993, TIBS 17, 89-93; Kumar et al., 1995, FASEB J., 9, 1183; Breaker, 1996, Curr. Op. Biotech., 7, 442; Breaker, 1997, Nature Biotech. 15, 427).
There are several reports that describe the use of a variety of in vitro and in vivo selection strategies to study structure and function of catalytic nucleic acid molecules (Campbell et al., 1995, RNA 1, 598; Joyce 1989, Gene, 82,83; Lieber et al., 1995, Mol Cell Biol. 15, 540; Lieber et al., International PCT Publication No. WO 96/01314; Szostak 1988, in Redesigning the Molecules of Life, Ed. S. A. Benner, pp 87, Springer-Verlag, Germany; Kramer et al., U.S. Pat. No. 5,616,459; Draper et al., U.S. Pat. No. 5,496,698; Joyce, U.S. Pat. No. 5,595,873; Szostak et al., U.S. Pat. No. 5,631,146).
The enzymatic nature of a ribozyme is advantageous over other technologies, since the effective concentration of ribozyme sufficient to effect a therapeutic treatment is generally lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme (enzymatic nucleic acid) molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base-pairing. Thus, it is thought that the specificity of action of a ribozyme is greater than that of antisense oligonucleotide binding the same RNA site.
The development of ribozymes that are optimal for catalytic activity would contribute significantly to any strategy that employs RNA-cleaving ribozymes for the purpose of regulating gene expression. The hammerhead ribozyme, for example, functions with a catalytic rate (k.sub.cat) of .about.1 min.sup.-1 in the presence of saturating (10 mM) concentrations of Mg.sup.2+ cofactor. However, the rate for this ribozyme in Mg.sup.2+ concentrations that are closer to those found inside cells (0.5-2 mM) can be 10- to 100-fold slower. In contrast, the RNase P holoenzyme can catalyze pre-tRNA cleavage with a k.sub.cat of .about.30 min.sup.-1 under optimal assay conditions. An artificial `RNA ligase` ribozyme (Bartel et al, supra) has been shown to catalyze the corresponding self-modification reaction with a rate of .about.100 min.sup.-1. In addition, it is known that certain modified hammerhead ribozymes that have substrate binding arms made of DNA catalyze RNA cleavage with multiple turnover rates that approach 100 min.sup.-1. Finally, replacement of a specific residue within the catalytic core of the hammerhead with certain nucleotide analogues gives modified ribozymes that show as much as a 10-fold improvement in catalytic rate. These findings demonstrate that ribozymes can promote chemical transformations with catalytic rates that are significantly greater than those displayed in vitro by most natural self-cleaving ribozymes. It is then possible that the structures of certain self-cleaving ribozymes may not be optimized to give maximal catalytic activity, or that entirely new RNA motifs could be made that display significantly faster rates for RNA phosphoester cleavage.
An extensive array of site-directed mutagenesis studies have been conducted with ribozymes such as the hammerhead, hairpin, hepatitis delta virus, group I, group II and others, to probe relationships between nucleotide sequence, chemical composition and catalytic activity. These systematic studies have made clear that most nucleotides in the conserved core of these ribozymes cannot be mutated without significant loss of catalytic activity. In contrast, a combinatorial strategy that simultaneously screens a large pool of mutagenized ribozymes for RNAs that retain catalytic activity could be used more efficiently to define immutable sequences and to identify new ribozyme variants.
Certain strategies to optimize reagents, such as the ribozymes, to down regulate the expression of a known target sequence have recently been reported:
Kramer et al., U.S. Pat. No. 5,616,459, describe a selection method for optimizing a hammerhead or a hairpin ribozyme by mutagenizing the "catalytic domain" of these ribozymes while keeping the binding arm sequence constant. Hammerhead or hairpin ribozymes optimal for cleaving a specific known target site are selected.
Roninson et al., U.S. Pat. No. 5,217,889, and Draper et al., U.S. Pat. No. 5,496,698, describe a method for selecting ribozymes capable of cleaving a known target sequence by fragmenting the DNA of the target gene, inserting the catalytic core of a known ribozyme into these DNA fragments, cloning these fragments into a vector, expressing these ribozymes in a cell and selecting for the vector encoding the optimal ribozyme.
Draper et al., U.S. Pat. No. 5,496,698, also describes a method for identifying ribozyme cleavage sites in a known RNA target by using ribozymes with randomized binding arms. Draper states on column 2, third full paragraph:
"Applicant provides an in vivo system for selection of ribozymes targeted to a defined RNA target. The system allows many steps in a selection process for desired ribozymes to be bypassed. In this system, a population of ribozymes having different substrate binding arms (and thus active at different RNA sequences) is introduced into a population of cells including a target RNA molecule. The cells are designed such that only those cells which include a useful ribozyme will survive, or only those cells including a useful ribozyme will provide a detectable signal. In this way, a large population of randomly or non-randomly formed ribozyme molecules may be tested in an environment which is close to the true environment in which the ribozyme might be utilized as a therapeutic agent." (Emphasis added)
Leiber et al., supra, describes a method for screening a known target RNA for accessible ribozyme cleavage sites. This method involves the incubation of a library of hammerhead ribozymes, with randomized binding arms, with the target RNA in vitro and identification of hammerhead ribozymes that cleave the target RNA. The selected ribozymes are then introduced into a cell to test their activity.
The references cited above are distinct from the presently claimed invention since they do not disclose and/or contemplate the nucleic acid molecules and the methods for target site selection and discovery of the instant invention.